Engineering success
We started with the idea of making a kit for detecting tau in tears to diagnose mild traumatic brain injury (mTBI). This led to us doing further research on mTBI and its biomarker. Noticeably, the tau protein was one of the biomarkers used to diagnose mTBI, and led to us thinking of a way to capture tau proteins in tears. However, before we proceeded to design our project, we conducted various interviews to a wide range of people to see if our kit would be an innovative enough idea, and how impactful our kit would become. Based on the interviews, we have received various inputs. For example, a health care worker implied that our kit would be cost efficient and time efficient (not only for the patient but for the healthcare worker as well), and it would be a kit that would provide a great impact on the public, and ice-hockey players. In another one of our interviews, a researcher suggested that we use aptamers in our project. This concept was new to most of the members, as aptamers was a relatively new concept. Thus, we conducted extensive research on aptamers as well as a new method called RCA-LAMP: a newly used method for amplification of nucleotide sequences. Aptamers are chosen as the novelty to this project, as it is a single strand of oligonucleotides that can be modified to the specific binding of our desired target. This gave us the idea to modify the aptamer to bind to the tau protein so that we could be able to detect possible minor traumatic brain injuries. In order to do so, we used the method RCA-LAMP. This method uses a padlock probe, p53 sequence (which serves as a recognition site for the padlock probe); and in our kit, a linker. The linker is used to connect the aptamer to the p53 sequence in the padlock probe, and create some distance between them. However, this raised some concerns, ‘would the linker possibly interfere with the binding between aptamer and tau protein?’. In order to verify whether an interference would occur if the linker is present and would disrupt the binding between the aptamer and tau protein, we modeled the structure of the aptamer and ran multiple simulations and programs to obtain the aptamer structure, and information regarding the binding between the aptamer and tau, along with the binding between the aptamer, tau, and linker. Based on the results, it is confirmed that the linker does not interfere with the binding between tau and aptamer. After this confirmation, we continued to design protocols that we would follow during the wet lab, as well as risk assessments of possible chemicals that would be used. First, we planned a protocol for tau protein transfection, agarose gel, SDS-PAGE, IMAC, and LAMP.
We added a plasmid containing the coding sequences for antibiotic resistance against kanamycin, hexa-histidine tag in fusion with the tau protein, and GFP, into the E.coli colonies. This means that we can use antibiotics to find the transfected cells, and due to the presence of GFP, the fusion protein would have a visible green color. As the fusion protein would contain a hexa-histidine tag, we used the IMAC method for protein purification. This would allow the binding between the magnetic bead and hexa-histidine tag. We then grew the bacteria in agar laden petri dishes containing the antibiotic Kanamycin to only get the transfected cells. The controlled variables are the petri dishes containing only the agar. After culturing the bacteria in wet culture, we lyzed them to acquire the proteins from the bacteria. The plasmid was sent off to a sequencing facility in Germany to verify our DNA sequence. We were later informed that the transfection occurred as we had hoped. The proteins were run on an SDS-PAGE apparatus to verify the size. We used nickel beads that bound to the his tag to clean out them via IMAC. We also used a nanodrop to get the concentration of the protein. Additionally, synthetic tears were made to mimic human tears in order to obtain more reliable results that are applied to the real world. Synthetic tears were used to run with LAMP.
The aptamer-LAMP setup was tested with tau and tears. To look at the aptamer binding, we combined the tau+aptamer complex and ran it on an agarose gel, and compared it to just pure aptamer, as the bound complex would have a higher mass and migrate shorter if bound to the verified tau, compared to the tau alone. However, we noticed that in our results, we could not detect the aptamer. Thus, LAMP was run again but this time to see if this would occur again. After the repetition, it was noticed that the aptamer did not show within the SDS-PAGE. Thus, we reviewed our protocol again to see if the design of the protocol would require trouble-shooting. This was done every week to ensure that the results we obtained would be as accurate and precise as possible.
Throughout the whole process, it has taught us to develop and hone our innovative skills, especially in the lab, as modification of the protocol would occur and we were required to figure out which part needs changing, and ‘why?’. So, every week the lab would discuss results that have been obtained from the lab. For example, the first week of lab, we had to culture the bacterias overnight. There was an issue that the tau protein was not present in the bacterias. Therefore, we analyzed the protocol to identify possible errors, and noticed that the incubation period could be a possible source of error, as it should have been left for longer. Thus, we cultured the bacteria again and incubated longer to see if there would be any effects. The new culture contained the tau protein that we expected. Furthermore, during the last few weeks of the lab, we noticed that our kit started amplification in the negative control. Therefore, all components used in RCA-LAMP were dissected to pinpoint a possible problem. We theorized that our primer dimerization could be the culprit for there to be no binding between the tau protein and aptamer, and could be the reasons for the false positive results. Thus, we conducted the experiment with a series of primer concentrations to see if the results would change and if it would become negative. But to no avail, the results still remained positive. Hence, we continued to see if there were other components in our kit that were possibly ‘incorrect’ or the reason for these false positives. However, further experiments would be required but we are positive that this issue could be resolved.